4.6 Article

A Combined Acceptor Photobleaching and Donor Fluorescence Lifetime Imaging Microscopy Approach to Analyze Multi-Protein Interactions in Living Cells

期刊

出版社

FRONTIERS MEDIA SA
DOI: 10.3389/fmolb.2021.635548

关键词

multiple protein-protein interactions; three-fluorophore FRET; 3-way FRET; acceptor photobleaching; FLIM

资金

  1. Deutsche Forschungsgemeinschaft [DFG INST 271/342-1, DFG BE 3246/6-1]
  2. European Regional Development Fund of the European Commission [W21029490]
  3. Martin-Luther-University Halle/Wittenberg [VAT DE 811353703]

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Protein-protein interaction studies provide insights into the formation of complexes, regulation of enzymatic activity, and information transfer within cells. FRET microscopy is a powerful tool for non-invasive investigation of these interactions, allowing for analysis of multiple interacting proteins simultaneously. By combining different FRET data acquisition and interpretation methods, it is possible to distinguish between different types of protein-protein interactions in living cells.
Protein-protein interaction studies often provide new insights, i.e., into the formation of protein complexes relevant for structural oligomerization, regulation of enzymatic activity or information transfer within signal transduction pathways. Mostly, biochemical approaches have been used to study such interactions, but their results are limited to observations from lysed cells. A powerful tool for the non-invasive investigation of protein-protein interactions in the context of living cells is the microscopic analysis of Forster Resonance Energy Transfer (FRET) among fluorescent proteins. Normally, FRET is used to monitor the interaction state of two proteins, but in addition, FRET studies have been used to investigate three or more interacting proteins at the same time. Here we describe a fluorescence microscopy-based method which applies a novel 2-step acceptor photobleaching protocol to discriminate between non-interacting, dimeric interacting and trimeric interacting states within a three-fluorophore setup. For this purpose, intensity- and fluorescence lifetime-related FRET effects were analyzed on representative fluorescent dimeric and trimeric FRET-constructs expressed in the cytosol of HEK293 cells. In particular, by combining FLIM- and intensity-based FRET data acquisition and interpretation, our method allows to distinguish trimeric from different types of dimeric (single-, double- or triple-dimeric) protein-protein interactions of three potential interaction partners in the physiological setting of living cells.

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